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COMPARATIVE ANALYSIS OF HIV-1 AND MLV 3D NUCLEAR DISTRIBUTION 1

Valentina Quercioli1, Cristina Di Primio1, Antonio Casini2, Lubbertus C.F. Mulder3, Lenard S. 2

Vranckx4, Doortje Borrenberghs4,5, Rik Gijsbers4, Zeger Debyser4, Anna Cereseto2# 3

4

1 Laboratory of Biology, Scuola Normale Superiore, 56124 Pisa, Italy 5

2 Laboratory of Molecular Virology, University of Trento, Centre for Integrative Biology, 38123 6

Trento, Italy 7

3 Department of Microbiology, 6 Global Health and Emerging Pathogens Institute, Icahn School of 8

Medicine at Mount Sinai, New York, NY, 10029, USA. 9

4 Laboratory of Molecular Virology and Gene Therapy, KU Leuven, 3000 Leuven, Flanders, 10

Belgium 11

5 Laboratory for Photochemistry and Spectroscopy, Department of Chemistry, KU Leuven, 12

Celestijnenlaan 200F, 3001 Leuven, Belgium. 13

14

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# Address correspondence to Anna Cereseto, Laboratory of Molecular Virology, CIBIO, Via 16

Sommarive 9, 38123, Trento, Italy. Phone: (+39) 0461 283282. Email: anna.cereseto@unitn.it 17

JVI Accepted Manuscript Posted Online 9 March 2016J. Virol. doi:10.1128/JVI.03188-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 18

Recent advances in fluorescence microscopy allow 3D analysis of HIV-1 pre-integration complexes 19

in the nuclei of infected cells. To extend this investigation to gamma-retroviruses, we engineered a 20

fluorescent Moloney murine leukemia virus (MLV) system, MLV-IN-EGFP. The comparative 21

analysis between lentiviral (HIV-1) and gamma-retroviral (MLV) fluorescent viral complexes in the 22

nuclei of infected cells revealed their different spatial distribution. This research tool has the 23

potential to reveal new insight in the nuclear biology of these retroviruses. 24

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To complete its replication cycle HIV-1 has to reach the nucleus of the infected cells where the viral 26

cDNA integrates into the host chromatin (1). Although the integration reaction and the targeted 27

chromatin regions have been well characterized, very little is known about the virus-cell 28

interactions taking place within the nuclear compartment. 29

The development of fluorescence imaging approaches to study HIV-1 early replication steps, has 30

significantly contributed to our knowledge of the HIV-1 life cycle. Indeed, fluorescence imaging 31

techniques allow the observation at a single particle level of HIV-1 interactions with the target cell 32

structures preserving their main 3D structural properties. Imaging techniques have revealed that 33

HIV-1 particles (2–5) and integrated proviruses (6), preferentially localize in the nuclear periphery, 34

which is consistent with the observation that peripheral chromatin, especially that near the nuclear 35

pore complexes, is favored for HIV-1 integration (7, 8). 36

Here we further analyze the 3D nuclear distribution of HIV-1 pre-integration complexes (PICs) in 37

relation to the expression of HIV-1 integrase co-factor LEDGF/p75 (for a recent review see 38

Debyser et al (9)). We also developed a new tool for the detection of fluorescent MLV which 39

allowed us to compare the 3D nuclear distribution of gamma-retroviral and lentiviral particles. 40

We set out to explore the role played by LEDGF/p75 in the 3D nuclear distribution of HIV-1 in the 41

nucleus during the early phases of infection. Using a technique that allows us to produce and track 42

fluorescent HIV-IN-EGFP PICs (2–4), we analyzed the virus localization with respect to the levels 43

of chromatin condensation as demarcated by the ectopic expression of the histone H2B fused to the 44

red fluorescent protein, H2B-RFP (2). Using the same procedures we previously established (2–4), 45

we analyzed the localization of HIV-IN-EGFP pre-integration complexes (PICs) in HeLa-H2B-RFP 46

cells stably silenced for LEDGF/p75 (10) (Fig. 1A). Figure 1B shows the preferential localization 47

of HIV-IN-EGFP PICs toward less condensed euchromatic regions characterized by low H2B 48

fluorescence. Statistical analysis using the non-parametric two-tailed Kolmogorov-Smirnov test 49

revealed no significant difference between the distribution of HIV-1 PICs in either LEDGF/p75 50

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knockdown or control cells (P=0.76 blue and pink curves). Conversely, HIV-PICs distribution 51

towards euchromatin in both cell lines significantly differs from the random region of interests 52

(ROIs) distribution in the same cells (grey and black curves from control and LEDGF/p75 53

knockdown cells respectively) (Fig. 1C). Analyses performed using an heterochromatin specific 54

marker (H3K9me3) confirmed a preferential localization of HIV-1 PICs in regions of the chromatin 55

with low intensity of H3K9me3 signals (Fig. 1D), which is similar to what we previously observed 56

in wild-type cells (2). Therefore, even though LEDGF/p75 is the main factor directing HIV-1 57

toward specific gene-rich regions (9, 11), it does not play a crucial role in the macro-localization of 58

HIV-1 viral complexes in the nucleus. 59

To confirm that the absence of an HIV-1 PICs relocalization phenotype in LEDGF/p75 knockdown 60

cells was not due to intrinsic limitations of the assay, the analysis was repeated in LEDGF/p75 61

knockdown cells stably expressing CBX-LEDGF(325-530) (12) (Fig. 1A). CBX-LEDGF(325-530) 62

is a chimeric LEDGF/p75 molecule engineered to contain an alternative chromatin-binding domain, 63

CBX1, and is reported to strongly relocalize HIV-1 integration toward heterochromatin (12–14). As 64

expected in these cells we observed that HIV-IN-EGFP PICs are randomly distributed (Fig 1E), 65

thus confirming that the HIV-1 imaging tool correctly detects the HIV-1 3D macro-localization. 66

Therefore, even though LEDGF/p75 knockdown generates a more random distribution of integrated 67

proviruses (9, 15–17), in the absence of this integrase co-factor HIV-1 PICs preserve their 68

localization toward subnuclear regions occupied by euchromatin. 69

To compare the nuclear localization of lenti- and gamma-retroviruses we developed MLV-IN-70

EGFP, a fluorescently labeled MLV. To produce fluorescently labeled viral particles a Moloney 71

MLV retroviral packaging vector, pMLV-gag-pol-EGFP, was generated (Fig. 2A). Fusion of EGFP 72

at the C-terminus of the MLV-Pol does not impair the association between IN and BRD4 (18–20) 73

as demonstrated by the co-immunoprecipitation experiment performed in 293T cells transfected 74

with the pMLV-gag-pol-EGFP construct (Fig. 2B). To further characterize the MLV-IN-EGFP, we 75

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tested the infectivity of the retroviral transfer vector pBabe-Puro (21) packaged with pMLV-gag-76

pol-EGFP (Fig. 2C) and pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G). These 77

experiments showed that the infectivity of MLV-IN-EGFP is similar to the one obtained with wild-78

type MLV (pBabe-Puro vector packaged with the parental phCMV-intron gag-pol) (Fig 2C). We 79

next analyzed the MLV-IN-EGFP viral complexes after infection of HeLa H2B-CFP (cyan 80

fluorescent protein) and found them to be localized both in the cytoplasm (Fig. 2D, upper panel) as 81

well as in the nuclear compartment as indicated by the 3 axis nuclear analysis (Fig. 2D, lower 82

panel). Twenty-four hours post infection an average of 3 MLV-IN-EGFP complexes per nucleus 83

were detected by using viral preparations made with an amphotropic MLV envelope (strain 4070A), 84

while with VSV-G pseudotyped virions we detected an average of 16 complexes per nucleus (Fig. 85

2E). Gammaretroviruses, such as MLV need the target cell to go through mitosis in order to 86

complete infection (22). This has been attributed to their inability to actively cross the nuclear 87

envelope (22). We thus tested the cellular localization of MLV-EGFP complexes in cells whose cell 88

cycle was blocked in G1 by aphidicolin treatment. As expected, MLV complexes were restricted to 89

the cytoplasmic compartment in aphidicolin treated cells, and nuclear complexes could not be 90

detected (Fig. 3A). As reported earlier (23), also in this case MLV-IN-EGFP particles were detected 91

associated to condensed chromatids in cells going through mitosis (Fig. 3B). 92

To compare the nuclear distribution of MLV complexes to that of HIV-1, the localization of MLV 93

was assessed with respect to the nuclear periphery and chromatin condensation. Unlike HIV-1, the 94

MLV complexes were homogenously distributed in the volume spanning from the periphery to the 95

center of the nuclei (Fig. 4A). Moreover, in cells marked with fluorescent H2B histones, the MLV 96

complexes did not show any preferential localization toward either more or less condensed regions 97

of the chromatin (Fig. 4B). To better analyze this aspect, the MLV distribution was analyzed in 98

cells immunostained with epigenetic markers of transcriptional repression (H3K9Me3) or activation 99

(H4K16Ac) (24). Also in this case, we could not observe any preferential localization of MLV with 100

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respect to either of these two markers (Fig. 4C-D). These results demonstrate that while HIV-1 101

PICs are mainly found in subnuclear compartments occupied by euchromatin and near the nuclear 102

periphery (2, 4), MLV PICs do not show any preferential localization. 103

Therefore, even though both HIV-1 and MLV prefer to integrate within transcription units (25) 104

associated with less condensed euchromatic regions, the subnuclear 3D macrodistribution of their 105

PICs differs significantly. This difference can probably be attributed to the different mechanisms by 106

which HIV-1 and MLV enter the nuclei of the infected cells: either through nuclear pore complexes 107

in HIV-1 or during mitosis and nuclear envelope disassembly in MLV. Based on this hypothesis 108

nucleoporins or other factors might be relevant for 3D micro-distribution. Alternatively, non-109

random distribution assumed by HIV-1 PICs might simply result from their position immediately 110

after transition through the nuclear pore complexes. In this scenario PIC distribution would be 111

determined by physical events rather than by any specific cellular factor. 112

The microscopy tool reported here, by allowing the analysis of the viral PIC nuclear distribution 113

during infection provides a set of complementary information to those obtained by linear 114

sequencing, thereby shedding light on new aspects of retroviral biology. 115

116

Acknowledgements: 117

This work was supported by grants from the EU FP7 (THINC, Health-2008-201032), by the 118

Provincia Autonoma di Trento (COFUND project, Team 2009 – Incoming). 119

phCMV-intron gag-pol is a gift from François-Loic Cosset, LVRTG, ENS de Lyon–U412 120

INSERM, Lyon, France. L.C.F.M. is supported by NIH-NIGMS grant R01 GM113886 121

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REFERENCES 123

1. Craigie R, Bushman FD. 2012. HIV DNA integration. Cold Spring Harb Perspect Med 124

2:a006890. 125

2. Albanese A, Arosio D, Terreni M, Cereseto A. 2008. HIV-1 pre-integration complexes 126

selectively target decondensed chromatin in the nuclear periphery. PloS One 3:e2413. 127

3. Francis AC, Di Primio C, Quercioli V, Valentini P, Boll A, Girelli G, Demichelis F, 128

Arosio D, Cereseto A. 2014. Second Generation Imaging of Nuclear/Cytoplasmic HIV-1 129

Complexes. AIDS Res Hum Retroviruses 30:717–726. 130

4. Cereseto A, Giacca M. 2014. Imaging HIV-1 nuclear pre-integration complexes. Methods 131

Mol Biol Clifton NJ 1087:47–54. 132

5. Burdick RC, Hu W-S, Pathak VK. 2013. Nuclear import of APOBEC3F-labeled HIV-1 133

preintegration complexes. Proc Natl Acad Sci U S A 110:E4780–4789. 134

6. Di Primio C, Quercioli V, Allouch A, Gijsbers R, Christ F, Debyser Z, Arosio D, Cereseto 135

A. 2013. Single-cell imaging of HIV-1 provirus (SCIP). Proc Natl Acad Sci U S A 110:5636–136

5641. 137

7. Marini B, Kertesz-Farkas A, Ali H, Lucic B, Lisek K, Manganaro L, Pongor S, Luzzati 138

R, Recchia A, Mavilio F, Giacca M, Lusic M. 2015. Nuclear architecture dictates HIV-1 139

integration site selection. Nature 521: 227-231. 140

8. Lelek M, Casartelli N, Pellin D, Rizzi E, Souque P, Severgnini M, Di Serio C, Fricke T, 141

Diaz-Griffero F, Zimmer C, Charneau P, Di Nunzio F. 2015. Chromatin organization at the 142

nuclear pore favours HIV replication. Nat Commun 6:6483. 143

on February 13, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

8

9. Debyser Z, Christ F, De Rijck J, Gijsbers R. 2015. Host factors for retroviral integration site 144

selection. Trends Biochem Sci 40:108–116. 145

10. Schrijvers R, De Rijck J, Demeulemeester J, Adachi N, Vets S, Ronen K, Christ F, 146

Bushman FD, Debyser Z, Gijsbers R. 2012. LEDGF/p75-independent HIV-1 replication 147

demonstrates a role for HRP-2 and remains sensitive to inhibition by LEDGINs. PLoS Pathog 148

8:e1002558. 149

11. Van Maele B, Busschots K, Vandekerckhove L, Christ F, Debyser Z. 2006. Cellular co-150

factors of HIV-1 integration. Trends Biochem Sci 31:98–105. 151

12. Gijsbers R, Ronen K, Vets S, Malani N, De Rijck J, McNeely M, Bushman FD, Debyser 152

Z. 2010. LEDGF hybrids efficiently retarget lentiviral integration into heterochromatin. Mol 153

Ther J Am Soc Gene Ther 18:552–560. 154

13. Silvers RM, Smith JA, Schowalter M, Litwin S, Liang Z, Geary K, Daniel R. 2010. 155

Modification of integration site preferences of an HIV-1-based vector by expression of a novel 156

synthetic protein. Hum Gene Ther 21:337–349. 157

14. Ferris AL, Wu X, Hughes CM, Stewart C, Smith SJ, Milne TA, Wang GG, Shun M-C, 158

Allis CD, Engelman A, Hughes SH. 2010. Lens epithelium-derived growth factor fusion 159

proteins redirect HIV-1 DNA integration. Proc Natl Acad Sci U S A 107:3135–3140. 160

15. Ciuffi A, Llano M, Poeschla E, Hoffmann C, Leipzig J, Shinn P, Ecker JR, Bushman F. 161

2005. A role for LEDGF/p75 in targeting HIV DNA integration. Nat Med 11:1287–1289. 162

16. Marshall HM, Ronen K, Berry C, Llano M, Sutherland H, Saenz D, Bickmore W, 163

Poeschla E, Bushman FD. 2007. Role of PSIP1/LEDGF/p75 in lentiviral infectivity and 164

integration targeting. PloS One 2:e1340. 165

on February 13, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

9

17. Shun M-C, Raghavendra NK, Vandegraaff N, Daigle JE, Hughes S, Kellam P, 166

Cherepanov P, Engelman A. 2007. LEDGF/p75 functions downstream from preintegration 167

complex formation to effect gene-specific HIV-1 integration. Genes Dev 21:1767–1778. 168

18. De Rijck J, de Kogel C, Demeulemeester J, Vets S, El Ashkar S, Malani N, Bushman FD, 169

Landuyt B, Husson SJ, Busschots K, Gijsbers R, Debyser Z. 2013. The BET family of 170

proteins targets moloney murine leukemia virus integration near transcription start sites. Cell 171

Rep 5:886–894. 172

19. Gupta SS, Maetzig T, Maertens GN, Sharif A, Rothe M, Weidner-Glunde M, Galla M, 173

Schambach A, Cherepanov P, Schulz TF. 2013. Bromo- and extraterminal domain 174

chromatin regulators serve as cofactors for murine leukemia virus integration. J Virol 175

87:12721–12736. 176

20. Sharma A, Larue RC, Plumb MR, Malani N, Male F, Slaughter A, Kessl JJ, Shkriabai 177

N, Coward E, Aiyer SS, Green PL, Wu L, Roth MJ, Bushman FD, Kvaratskhelia M. 178

2013. BET proteins promote efficient murine leukemia virus integration at transcription start 179

sites. Proc Natl Acad Sci U S A 110:12036–12041. 180

21. Morgenstern JP, Land H. 1990. Advanced mammalian gene transfer: high titre retroviral 181

vectors with multiple drug selection markers and a complementary helper-free packaging cell 182

line. Nucleic Acids Res 18:3587–3596. 183

22. Roe T, Reynolds TC, Yu G, Brown PO. 1993. Integration of murine leukemia virus DNA 184

depends on mitosis. EMBO J 12:2099–2108. 185

on February 13, 2018 by guest

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nloaded from

10

23. Elis E, Ehrlich M, Prizan-Ravid A, Laham-Karam N, Bacharach E. 2012. p12 tethers the 186

murine leukemia virus pre-integration complex to mitotic chromosomes. PLoS Pathog 187

8:e1003103. 188

24. Cattoglio C, Pellin D, Rizzi E, Maruggi G, Corti G, Miselli F, Sartori D, Guffanti A, Di 189

Serio C, Ambrosi A, De Bellis G, Mavilio F. 2010. High-definition mapping of retroviral 190

integration sites identifies active regulatory elements in human multipotent hematopoietic 191

progenitors. Blood 116:5507–5517. 192

25. Mitchell RS, Beitzel BF, Schroder ARW, Shinn P, Chen H, Berry CC, Ecker JR, 193

Bushman FD. 2004. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target 194

site preferences. PLoS Biol 2:E234. 195

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FIGURES: 198

Figure 1: LEDGF/p75 is dispensable for the localization of HIV-1 complexes in nuclear 199

regions occupied by euchromatin. (A) Western blot analysis of HeLa-H2B-RFP LEDGF/p75 200

knockdown cells or LEDGF/p75 knockdown cells trans-complemented with CBX-LEDGF(325-201

530). Upper panel: Western blot developed with antibodies anti-LEDGF/p75 (Bethyl Laboratories, 202

Inc.); bottom panel: Western blot developed with an anti-GAPDH antibody to control protein 203

loading. (B) H2B-RFP fluorescence intensity distribution frequency for HIV-IN-EGFP PIC ROIs 204

(blue striped bars) and for Random ROIs (grey bars) in LEDGF/p75 knockdown cells. (C) 205

Distribution of cumulative probabilities plotted for HIV-IN-EGFP PIC ROIs or Random (Rnd) 206

ROIs toward H2B-RFP fluorescence in LEDGF/p75 knockdown (LEDGF KD) or control cells 207

(CTRL). Reported P values are obtained by the Kolmogorov-Smirnov test (n=100). (D) H3K9Me3 208

fluorescence intensity distribution frequency for HIV-IN-EGFP PIC ROIs (red striped bars) and for 209

Random ROIs (grey bars) in LEDGF/p75 knockdown cells. In the inset the distributions of 210

cumulative probabilities are plotted for PIC ROI (red) and Random ROIs (grey). P value is obtained 211

by the Kolmogorov-Smirnov test (n=100). (C) H3K9Me3 fluorescence intensity distribution 212

frequency for PIC ROIs (red striped bars) and for Random ROIs (grey bars) in LEDGF/p75 213

knockdown cells expressing CBX-LEDGF(325-530). In the inset the distributions of cumulative 214

probabilities are plotted for PIC ROI (red) and Random ROIs (grey). P value is obtained by the 215

Kolmogorov-Smirnov test (n=100). A.u.= arbitrary units. 216

Figure 2: MLV-IN-EGFP visualization system. (A) Schematic representation of the plasmid used 217

to produce MLV-IN-EGFP particles. The construct was prepared as follows: the 3’ terminal end of 218

MLV pol was amplified using the primers MLV pol 5’: 219

GATCCTCGAGCTATAGAAAATTCATCACCC and MLV pol 3’: 220

GATCAGATCTCCGGGGGCCTCGCGGGTTAAC, using as template DNA the phCMV-intron-221

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gag-pol plasmid (a gift from François-Loic Cosset), and cloned into the BglII site of the pEGFP-N1 222

plasmid (Clontech). Next, a SnaBI-XmaI fragment encompassing the beta-globin intron and MLV 223

gag-pol from phCMV-intron gag-pol was cloned into the SnaBI-XmaI of the pEGFP-N1 containing 224

the 3’ end of MLV pol. (B) HEK 293T cells transfected with the pMLV-gag-pol-EGFP or control 225

(pCMV) plasmids were immunoprecipitated with anti-EGFP antibodies (IP-EGFP) and analyzed by 226

western blot with anti-BRD4 antibodies (Bethyl Laboratories, Inc.) (upper panel) or anti-EGFP 227

antibodies (lower panel). Crude lysates (Input) were loaded as controls. (C) MLV-IN-EGFP viral 228

supernatants were produced by transfection of 5×106 HEK 293T cells with 20 µg of pBabe-Puro, 10 229

µg of pMLV-gag-pol-EGFP (or phCMV-intron-gag-pol) and 5 µg of pVSV-G using the PEI 230

reagent. Supernatants were collected after 48 hrs and filtered through a 0.45 µm pore size filter. 231

Infectivity of MLV-IN-EGFP was measured by counting the number of HeLa colonies resistant to 232

puromycin 2 weeks post infection with 2.87 reverse transcriptase units (RTU) (measured by PCR-233

enhanced reverse transcriptase-PERT) assay (3)) and approximately corresponding to MOI=1. (D) 234

HeLa cells expressing H2B-CFP and infected with MLV-IN-EGFP (2.87 RTU) in a 3D image 235

(upper panel) or a derived confocal section (bottom panel). (E) Number of MLV-IN-EGFP 236

complexes in the nuclei of HeLa cells 24 hours post infection. MLV-A and VSV-G indicate the 237

envelopes used to produce the MLV-IN-EGFP viral particles. 238

Figure 3: Nuclear detection of MLV complexes. (A) Confocal section obtained from HeLa cells 239

infected with MLV-IN-EGFP particles (green spots) and immunostained with lamin A/C (blue) (left 240

image). Confocal section obtained from HeLa cells treated with 5 µM aphidicolin, infected with 241

MLV-IN-EGFP particles (green spots) and immunostained with lamin A/C (blue) (right image). 242

Image analysis of fluorescent nuclear PICs is described in (2-4). PIC/nucleus values indicate the 243

average number of nuclear MLV-IN-EGFP complexes detected in n=90 cells from both conditions. 244

(B) Visualization of MLV-EGFP complexes attached to chromatids (green arrows). 245

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Figure 4: 3D nuclear distribution of MLV complexes. (A) Distance of intranuclear MLV 246

complexes from the lamin A/C staining. (B) H2B-CFP fluorescence intensity distribution frequency 247

for MLV ROIs (black striped bars) and for Random ROIs (grey bars). In the inset the distributions 248

of cumulative probabilities are plotted for MLV ROIs (black) and Random ROIs (grey). (P=0.35, 249

Kolmogorov-Smirnov test) (n=400). (C) H3K9Me3 fluorescence intensity distribution frequency 250

for MLV ROIs (black striped bars) and for Random ROIs (grey bars). In the inset the distributions 251

of cumulative probabilities are plotted for MLV ROIs (black) and Random ROIs (grey). (P=0.33, 252

Kolmogorov-Smirnov test) (n=270). (D) H4K16ac fluorescence intensity distribution frequency for 253

MLV ROIs (black striped bars) and for Random ROIs (grey bars). In the inset the distributions of 254

cumulative probabilities are plotted for MLV ROI (black) and Random ROIs (grey). (P>=0.96, 255

Kolmogorov-Smirnov test) (n=100). A.u.= arbitrary units. 256

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